Nitrification is the microbial-mediated transformation of ammonium (NH4+) into nitrate (NO3-). Many plant species depend on the availability of NO3- as the main source of nitrogen (N). On the other hand, because NO3- is highly mobile in the soil profile, its excess concentration can cause environmental pollution. Nitrification can be estimated at the process level, but with the development of molecular techniques it is also possible to estimate the abundance of nitrifying bacteria in the soil. Hence, in this chapter we describe the procedure for detection and quantification of nitrifying bacteria in soil samples using real-time quantitative polymerase chain reaction (PCR).